Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

Sviridov, Denis; Meilinger, Bonnie; Drake, Steven K.; Hoehn, Gerard; Hortin, Glen L.

doi:10.1373/clinchem.2005.057323

Cited by 71 publications

(75 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…When albumin does denature at extremely high temperature or pH, it aggregates irreversibly; this leads to precipitation of albumin rather than additional forms in solution (25 ). Some dimerization of albumin is observed with many preparations, including purified plasma albumin (21,25 ), but as in previously reported studies, we found albumin dimers to be minor components in urine. Aggregation of albumin with other components such as the Tamm-Horsfall protein would result in elution in size-exclusion chromatography as a much larger complex, and that has not been observed (15,21 ).…”

Section: Discussionsupporting

confidence: 88%

“…This bias has been hypothesized to result from modified forms of urinary albumin that are detected by the size-exclusion assay but not by immunoassays. Our laboratory, how-ever, determined that size-exclusion chromatography does not resolve albumin from a number of other urinary proteins and is an assay for a mixture of albuminsized molecules (ranging from about 40 000 to 80 000 Da) rather than an assay for albumin (21 ). This finding offers an alternative explanation for the difference between immunoassay and size-exclusion chromatography but does not completely rule out some contribution from modified forms of albumin with decreased immunoreactivity.…”

confidence: 97%

“…A second source of uncertainty in urine albumin measurement is the lack of agreement of immunoassays for urine albumin with a size-exclusion chromatographic assay, which yields substantially higher values than immunoassays for many specimens from healthy individuals or diabetes patients (15)(16)(17)(18)(19)(20)(21)(22). This bias has been hypothesized to result from modified forms of urinary albumin that are detected by the size-exclusion assay but not by immunoassays.…”

confidence: 99%

See 2 more Smart Citations

Reactivity of Urinary Albumin (Microalbumin) Assays with Fragmented or Modified Albumin

Sviridov¹,

Drake

Hortin³

2008

Clinical Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Controversy exists regarding occurrence and measurement of structural variants of albumin in urine. In this study, we examined cross-reactivity of in vitro modified albumins in assays for urine albumin (microalbumin). Methods: We analyzed albumin modified by reagents, trypsin, or physical treatments or differing in primary sequence (animal albumins) with an immunoturbidimetric assay (Beckman LX20) using goat antiserum and a competitive immunoassay (Siemens Immulite) using a monoclonal antibody. We assessed occurrence of albumin fragments in urine by use of Western blotting of 24 specimens. Results: Chemical modification, modest sequence substitution (gorilla albumin), or cleavage of albumin by cyanogen bromide (CNBr) had little effect on reactivity in the LX20 assay. Albumin extensively cleaved with trypsin retained partial reactivity. The Immulite assay generally was affected more severely by albumin modifications and sequence changes. Western blots of fresh urine specimens or specimens stored at −80 °C showed little albumin fragmentation, but some specimens stored for 3 years at −20 °C had extensively fragmented albumin that was detected by the LX20 but not the Immulite assay. Conclusions: Nearly equivalent reactivity of intact albumin and CNBr fragments in the immunoturbidimetric assay indicates reactivity of antibodies with multiple epitopes throughout albumin. Therefore, it is difficult to abolish reactivity of albumin in this type of urine albumin assay. Differential sensitivity of 2 assays to albumin modification identifies a potential source of assay nonequivalence in measuring urinary albumin, particularly for specimens stored at −20 °C.

show abstract

Section: Discussionsupporting

confidence: 88%

confidence: 97%

confidence: 99%

See 1 more Smart Citation

Reactivity of Urinary Albumin (Microalbumin) Assays with Fragmented or Modified Albumin

Sviridov¹,

Drake

Hortin³

2008

Clinical Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…This observation has lead to a controversial hypothesis that sizeexclusion chromatography detects a form of albumin that is not detected by immunoassay (114, 116 -118 ). This hypothesis has been questioned on the basis of the documented reactivity of polyclonal antisera with multiple antigenic sites in albumin (82,113 ). Moreover, the results of the size-exclusion method included other molecules of approximately the same size as albumin, including several urinary proteins (82 ).…”

Section: Current Routine Methods For Measuring Urine Albumin Routine mentioning

confidence: 99%

Current Issues in Measurement and Reporting of Urinary Albumin Excretion

et al. 2009

Self Cite

View full text Add to dashboard Cite

BACKGROUND: Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. CONTENT:The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the error-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences have been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no reference materials for either analyte in urine. The recommended reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethnicity) nor the continuous increase in risk related to albumin excretion. DISCUSSION:Clinical needs have been identified for standardization of (a) urine collection methods, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

show abstract

“…58 Its separation is based on differences in the sizes or weights of the analytes, which govern their access to the pore beads packed in a column. In general, the smaller analytes can enter the pores more easily and therefore spend more time in these pores, increasing their retention time.…”

Section: Size-exclusion Chromatographymentioning

confidence: 99%